ABSTRACT Proximal-to-distal peristaltic contractions of the upper urinary tract (UUT) smooth muscle coat propel waste from the kidney to the bladder. Defects in the peristaltic process are highly prevalent and clinically significant. For example, impaired urine outflow from the kidney causes pressure mediated dilation of renal tissues, or hydronephrosis. Hydronephrosis is the most commonly observed abnormality in children, detected in 1% of newborns, and is a leading cause of pediatric kidney failure. The overall goal of this project is to better understand the normal physiology and pathophysiology of the UUT. Indeed, despite the high morbidity associated with urinary tract dysfunctions, the mechanisms underlying renal pacemaker activity that triggers UUT peristalsis have remained elusive. To study this process, we have developed novel live imaging techniques to record the propagation of electrical and contractile excitation throughout the intact UUT. Results of our studies have revealed that hyperpolarization activate cation (HCN) channels are highly expressed and localized to renal pacemaker tissues of the murine UUT. HCN channel inhibition abolishes UUT pacemaker activity, and results in a loss of coordinated peristalsis. Instead of the proximal-to-distal contractile and electrical excitation observed in control UUTs, HCN inhibited explants exhibit near-simultaneous electrical activation throughout the UUT and twitch-like contractile activity. Thus, we have demonstrated ex-vivo that HCN+ cells of the UUT are renal pacemakers that set the origin and coordinate UUT peristalsis. Moreover, we have recently discovered that HCN channel expression is conserved to renal pacemaker tissues of the porcine and human urinary tracts, which share a unique anatomy and physiology. In Aim 1 of this proposal we will use a novel mouse model of hydronephrosis that lacks HCN+ cells in the UUT. We will use the live imaging techniques we have developed to determine if loss of HCN+ cells in vivo results in aberrant UUT peristalsis that underlies hydronephrosis. Aim 1 will also include mechanistic studies to begin to elucidate the transcriptional networks regulating HCN+ pacemakers. For Aim 2, we have recently developed a novel explant system to directly visualize the electrical and contractile properties of peristalsis in the porcine UUT. We will use this explant system to determine if HCN channel conductance is required for coordinated UUT peristalsis in a close homolog to humans. Results of these studies will provide much needed insight into the mechanisms underlying normal and aberrant UUT peristalsis in both lower-order and higher-order mammalian species. Long term translational implications of the studies include the development of novel treatments and diagnostics for uropathies such as hydronephrosis.